Assessment of Chinese medicinal herb metabolite profiles by UPLC-MS-based methodology for the detection of aristolochic acids

In this study, aristolochic acid in different herbal medicines containing a mixture of varying herb species was identified through fingerprint pattern similarities. Aristolochic acid I and II are nephrotoxic compounds naturally present in the Aristolochia plant species that are commonly used in Chinese herbal medicines. Twenty-four commercially available herbal formulations were extracted into an aqueous solution and injected into a UPLC-MS system. All the samples were analysed by multiple reaction monitoring (MRM) to check for the presence of aristolochic acids I and II. The same samples were then fingerprinted using two different gradient methods and the chromatograms deconvoluted into retention time (RT) and masses for the chemicals present taking concentration into account. Statistical analysis of this data revealed that samples were highly heterogeneous, and that the main differences between the preparations were concentrations of polar compounds. A model was constructed where the samples could be separated into two groups differentiated by the presence of the two forms of aristolochic acid.     

Detecting aristolochic acids in herbal remedies by liquid chromatography/serial mass spectrometry

Targeted liquid chromatography/serial mass spectrometry (LC/MS/MS) analysis, using a quadrupole ion-trap mass spectrometer, permitted the detection of aristolochic acids I and II in crude 70% methanol extracts of multi-component herbal remedies without any clean-up or concentration stages. The best ionisation characteristics were obtained using atmospheric pressure chemical ionisation (APCI) and by including ammonium ions in the mobile phase. Limits of detection for aristolochic acids were influenced by the level of interference created by other components in the sample matrix. They were determined to be between 250 pg and 2.5 ng on-column within a matrix containing compounds extracted from 2 mg of herbal remedy. With a herbal remedy that only permitted the higher limit of detection, this sensitivity was sufficient to detect the aristolochic acids extracted from 0.1% dry weight of Aristolochia manshuriensis included in the preparation.     

Alkaloids from Aristolochia manshuriensis (Aristolochiaceae)

From the ethanol extract of the stems of Aristolochia manshuriensis, two new alkaloids, namely manshurienine A ( 1 ) and manshurienine B ( 2 ), were isolated together with six known compounds, i.e., aristoloside ( 3 ), aristolochic acid I ( 4 ), aristolochic acid II ( 5 ), aristolic acid II ( 6 ), aristolochic acid IVa ( 7 ), and aristolochic acid IIIa ( 8 ). The structures of 1 and 2 were elucidated on the basis of spectroscopic methods, mainly by using ¹H‐and ¹³C‐NMR spectroscopy, and the known compounds 3 – 8 were identified by comparing their physical and spectroscopic data with those of authentic data reported in the literature.     

Evaluation of Chinese medicinal herbs fingerprinting by HPLC-DAD for the detection of toxic aristolochic acids

Aristolochic acids are known to contribute to various renal disorders; therefore, expanding the availability of analytical methodology to detect these compounds is important in order to assess the quality of Chinese herbal medicines in which they can be found. Twelve medicinal herbal samples were procured from various sources and extracted in duplicate prior to a "fingerprint" analysis using conventional HPLC-DAD. Multivariate analysis was performed on the entire chromatographed fingerprints. The resulting output was a partial least-square discriminant analysis model, which was able to evaluate the potential presence of aristolochic acids I and II as well as providing an individual herbal "fingerprint". The results of this study provide evidence that the presence of aristolochic acids contained within certain herbal extractions could be detected using a simple method, although some limitations apply to this method for quality control, since newly detected samples for aristolochic acid (positives) will need further confirmation with purity checks or MS hyphenation.     

Über die Isolierung und Charakterisierung von vier neuen Aristolochiasäuren (aus Aristolochia clematitis L.)

Zusammenfassung In den sauren Anteilen des Wurzelextraktes von Aristolochia clematitis L. konnten neben den bekannten Aristolochiasäuren-I und-II geringe Mengen von vier neuen ähnlichen Säuren nachgewiesen werden. Durch Methylierung, Äthylierung und Decarboxylierung des Säuregemisches und Trennung der Umsetzungsprodukte mit Hilfe der Säulen- und der Dünnschichtchromatographie konnten Abbauprodukte aller vier Säuren isoliert und charakterisiert werden. Massenspektrometrische Untersuchungen, IR-Spektroskopie und chemische Methoden zeigten, daß die neuen Verbindungen den Aristolochiasäuren-I und-II weitgehend entsprechen. Zwei Verbindungen sind phenolisch, die beiden anderen deren Methyläther.

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In addition to the already known aristolochic acids I and II, small amounts of four new acids of this class have been detected in the acidic fractions of extracts from the roots of Aristolochia clematitis L. Derivatives of all four acids have been isolated by methylation, ethylation and decarboxylation of the mixture of acids, and separation of the products by column and thin layer chromatography. Mass spectrometric investigation, IR spectra and chemical methods showed the new compounds to be very similar to the aristolochic acids I and II. Two of the compounds are phenols and the other two the corresponding methyl ethers.

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Herrn Prof. Dr.O. Hromatka zum 60. Geburtstag gewidmet.     

Rapid determination of aristolochic acids I and II in herbal products and biological samples by ultra-high-pressure liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs) are a mixture of structural-related compounds, in which aristolochic acid I (AA I) and aristolochic acid II (AA II) are reported to be correlated with Aristolochic acid nephropathy (AAN). In this work, a rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine AA I and AA II in herbal products and biological fluids. By using gradient elution with a mobile phase composed of a mixture of 10 mM ammonium formate buffer (pH 3.0) and acetonitrile, AAs could be determined within 10 min. Under optimum UHPLC-MS/MS conditions, the limit of detections was 0.14 and 0.26 ng mL⁻¹ for AA I and AA II, respectively. Run-to-run repeatability and intermediate precision of peak area for AA I and AA II were less than 5.74% relative standard deviation (RSD). Accuracy was tested by spiking 10, 100 and 1000 ng mL⁻¹ in rat serum and the recoveries were within 76.5-92.9%. Matrix effects were within 78.8-127.7%. The developed method was successfully applied to determine AA I and AA II in several herbal products and to investigate their pharmacokinetic behavior in female Wister rats. The result shows that the developed UHPLC-MS/MS method is efficient, sensitive, and accurate for the determination of AA I and AA II in herbal products and biological samples.     

Selective analysis of aristolochic acid I in herbal medicines by dummy molecularly imprinted solid‐phase extraction and HPLC

In this study, surface molecularly imprinted polymers were prepared as the selective sorbents for separation of aristolochic acid I in herbal medicine extracts by a facile approach. A less toxic dummy template, ofloxacin, was used to create specific molecule recognition sites for aristolochic acid I in the synthesized polymers. The polymers were characterized by Fourier‐transfer infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, elemental analysis, and nitrogen adsorption–desorption test. The adsorption capacity was calculated using adsorption kinetics, selectivity, and recycling experiments. The obtained polymers exhibited high thermostability, fast equilibrium time, and excellent binding ability. Subsequently, the polymers applied as the solid‐phase extraction absorbent was proposed and used for the enrichment and analysis of aristolochic acid I in herbal plants. The result showed that the aristolochic acid I was enriched up to 16 times after analysis by using high‐performance liquid chromatography. The good linearity for aristolochic acid I was obtained in the range of 0.1–200 μg/mL (R ² = 0.9987). The recovery and precision values were obtained (64.94–77.73%, RSDs% ≤ 0.8%, n  = 3) at three spiked concentration levels. This work provided a promising method for selective enrichment, extraction, and purification of aristolochic acid I from complex herbal plants.     

Study on separation of aristolochic acid I and II by micellar electrokinetic capillary chromatography and competition mechanism between SDS and beta-cyclodextrin

In this study, a rapid MEKC method using 40 mM sodium borate buffer containing 50 mM SDS as surfactant was developed for the analysis of aristolochic acid (AA) in Aristolochia plants. Baseline separation of AA-I and AA-II was achieved within 3 min with high separation efficiency, satisfactory sensitivity, repeatability, and recovery. Resolution between AA-I and AA-II is above 5 and great performance with higher than 200,000 theoretical plate numbers was obtained. The detection limits (based on 3 S/N) were both 1.0 microg/mL. Two kinds of AA in 35 herbal samples of Aristolochia plants were successfully determined. The competition mechanism between beta-CD and SDS was also investigated by changing the content ratio of beta-CD and SDS.     

Isolation of aristolochic acid I from herbal plant using molecular imprinted polymer composited ionic liquid‐based zeolitic imidazolate framework‐67

Aristolochic acid I is a toxic compound found in the genus of Aristolochia plants, which are commonly used as herbal cough treatment medicines. To remove the aristolochic acid I in extract efficiently and selectively, a molecularly imprinted polymer composed of ethylimidazole ionic liquid‐based zeolitic imidazolate framework‐67 was synthesized and used as the adsorbent. Under the conditions optimized by the software design expert, the sorbent showed highest adsorption amount of 34.25 mg/g in methanol/water (95:5, v/v) at 39°C for 138 min. The sorbent was then applied to solid phase extraction to isolate aristolochic acid I from the extract of the herbal plant Fibraurea Recisa Pierre. 0.043 mg/g of aristolochic acid I was obtained after the loading, washing, and elution processes. The limit of detection of 2.41 × 10^?5 mg/mL and good recoveries provided evidence for the accuracy of this method.     

Die Konstitution der Aristolochiasäuren III und IIIa

Zusammenfassung Es wurde die Konstitution der Aristolochiasäure-III und der Aristolochiasäure-IIIa durch die Synthese von Abbauverbindungen bestimmt.AS-IIIa ist die 3,4-Methylendioxy-6-hydroxy-10-nitrophenanthren-1-carbonsäure,AS-III der Methyläther vonAS-IIIa.AS-IIIa ist mit der Aristolochiasäure-C (aus Aristolochia debilis)³ identisch.

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The constitutions of aristolochic acid-III and of aristolochic acid-IIIa have been established by the synthesis of degradation products. Aristolochic acid-IIIa is 3,4-methylenedioxy-6-hydroxy-10-nitrophenanthrene-1-carboxylic acid, aristolochic acid-III its methyl ether. Aristolochic acid-III a has been found to be identical with aristolochic acid-C fromAristolochia debilis ³.

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Mit 2 Abbildungen     

Simultaneous determination of five aristolochic acids and two aristololactams in Aristolochia plants by high-performance liquid chromatography

An HPLC method was developed for the simultaneous determination of five aristolochic acids (AAs) and two aristololactams (ALs) in the following six Chinese drugs derived from Aristolochia species. Samples were analyzed on a C(18) column with acetonitrile and 3.7 mm phosphoric acid buffer gradient elution, detected at 260 nm. Assay was linear over the range (microg/mL) 0.386-38.6 for aristolochic acid Va, 0.632-63.2 for aristolochic acid IVa, 0.200-20.0 for 9-hydroxy aristolochic acid I, 0.352-35.2 for aristololactam II, 0.296-29.6 for aristolochic acid II, 0.274-27.4 for aristololactam I and 3.12-312 for aristolochic acid I. Average recoveries (%) of samples were 102.0, 95.9, 99.2, 102.2, 97.2, 97.1 and 97.8 for these seven constituents, respectively. The detection limit and retention time for the seven constituents ranged from 10.0 to 15.8 ng/mL and from 12 to 21 min. As a result of drug determination, contents (in mg/g) were as follows: AA-I, 0.69-1.77; AA-II, 0.02-0.18; 9-OH AA-I, 0.04-0.12; AA-IVa, 0.76-3.36; AA-Va, 0.04-0.31; AL-I, 0.07-0.36; and AL-II, 0.01-0.09 in Madouling; AA-I, 0.03-0.41; AA-II, 0.01-0.11; 9-OH AA-I, 0.00-0.60; AA-IVa, 0.00-0.77; AA-Va, 0.00-0.14; and AL-I, 0.00-0.04 in Tianxianteng; AA-I, 1.19-4.71; and AA-II, 0.24-1.69 in Qingmuxiang; AA-I, 2.79-5.48; AA-II, 1.06-1.86; 9-OH AA-I, 0.01-0.09; AA-IVa, 0.38-0.69; AA-Va, 0.00-0.61; AL-I, 0.00-0.02; and AL-II, 0.00-0.02 in Bei-madouling-gen; AA-I, 0.64-4.23; AA-II, 0.06-0.40; and AA-IVa, 0.08-0.25; in Guangfangji; and AA-I, 1.88-9.72; AA-II, 0.26-1.88; and AA-IVa, 0.09-0.52 in Guanmutong. The other constituents were not detected in Tianxianteng, Qingmuxiang, Guangfangji and Guanmutong.     

The Constituents of the Stem and Roots of Aristolochia foveolata

One new aristolactam glucoside: aristolactam-Ia-N-D-glucoside together with seventeen known compounds were isolated from the fresh stems and roots of Aristolochia foveolata. Their structures were determined by spectral and chemical methods. Among seven aristolochic acid derivatives, five were determined to be salt forms by IR and ¹H NMR methods.     

Enforcement of the ban on aristolochic acids in Chinese traditional herbal preparations on the Dutch market

In traditional chinese medicine several Aristolochia species are used. Aristolochia spp. contain a mixture of aristolochic acids (AAs), mainly AA I and AA II which are nephrotoxicants and carcinogens. After AA-related nephropathy (AAN) and urothelial cancer were described in female patients in Belgium following intake of AA-contaminated herbal preparations, herbs with AAs were prohibited worldwide. Confusing nomenclature can cause AA contamination of certain Chinese traditional herbal preparations (THPs). Here we report the results of investigations by the Dutch Food and Consumer Product Safety Authority (VWA) into the presence of AAs in THPs sampled on the Dutch market using a liquid-chromatography–-mass spectrometry method. Between 2002 and 2006 we sampled 190 Chinese THPs using recent information on Chinese THPs potentially containing AAs. AA I was found in 25 samples up to a concentration of 1,676 mg/kg. AA II was also found in 13 of these samples up to 444 mg/kg. All 25 positive samples including Mu Tong, Fang Ji, Tian Xian Teng and Xi Xin were part of a group of 68 THPs identified as possibly containing AAs. In a worst-case scenario, use of a sample of Mu Tong with the highest AA content over a 7-day period would result in the same intake levels of AAs which significantly raised the cancer risk in the Belgian AAN cases. Our results show that contaminated THPs still can be found on the market following worldwide publicity. Therefore, it can be concluded that testing of possibly AA-contaminated THPs is still essential. Figure Various Chinese Herbs     

Phase diagrams and phase structure of theN-octanoyl-L-glutamic acid oligomer benzyl esters-CHCl3 systems

N-Octanoyl-L-glutamic acid oligomer benzyl esters (residue number,N=1–4, 6, 8, and 12) have been synthesized. For the solid samples ofN=3–12, x-ray powder diffraction pattern and vibrational spectroscopic measurements have led to the assumption of a -sheet structure. For the CHCl₃-solutions of theN-octanoyl tetramers, hexamers, and octamers, the phase diagram consists of three regions (I, II, and III). Region I is an isotropic phase, in which the aggregate structure strongly depends upon concentration, and region II is a lyotropic liquid crystal area. Region III is a two-phase area in which regions I and II coexist. In the case of the trimer solutions, it consists of two regions (I and II). For the oligomers withN=3–12 in region I, it was assumed that micellization induces preferential stabilization of the -sheet structure depending on the concentration. Further stabilization of the -sheet structure, was found to occur in region II. For the hexamer, octamer, and dodecamer in CHCl₃, results from light-scattering measurements have led to an estimate of the apparent weight-average molecular weights and aggregation numbers of the micelle in region I.     

Determination of aristolochic acids in medicinal plant and herbal product by liquid chromatography-electrospray-ion trap mass spectrometry

This paper describes a liquid chromatography-electrospray-ion trap mass spectrometry (LC-ES-ITMS) method for the determination of aristolochic acid I and II (AA-I and AA-II) in medicinal plants and Chinese herbal remedies. A reversed phase C₁₈ column with gradient elution was utilized. The effects of mobile phase additives, acetic acid and ammonium acetate, on LC separation and ES ionization were investigated. For both AA-I and AA-II, the [M+NH₄]⁺ ion was found to be the precursor ion for target MS/MS analysis. The MS/MS product ion, [M+H−44]⁺, was used for the quantitative measurement of AA-I and AA-II. The linearity was good from 0.03 to 5 μg ml⁻¹ and good correlation (r²=0.999) over the range examined was determined for both AA. The detection limit based on a signal-to-noise ratio of three was 0.012 and 0.015 μg ml⁻¹ for AA-I and AA-II, respectively. Various Chinese herbal remedies obtained from renal failure patients and medicinal plants were examined by this newly developed method.     

Determination of Aristolochic Acid I and II in Traditional Chinese Medicine by HPCE with Label Free Intrinsic Imaging

Aristolochic acids (AAs) I and II are the main bioactive ingredients in most Aristolochia plants, which are used for dietary supplements, slimming pills and Traditional Chinese Medicines (TCMs). Excessive ingestion of AAs can lead to serious nephropathy and has also been discovered to cause mutagenicity and carcinogenicity. Therefore, quantitative analysis and quality control for medicines containing AA’s are of great importance. The analysis of aristolochic acid I and II in TCM was achieved successfully in less than 5 min by capillary electrophoresis with label free intrinsic imaging (LFII) and UV detection at λ₂₁₄ nm. The background electrolyte was deltaDOT’s TCM running buffer containing sodium tetraborate and β-cyclodextrin at pH 9.7. The results show very good resolution, precision and short analysis time with a detection limit of 1.23 × 10^?7 and 8.83 × 10^?8 mol L^?1 for AAI and AAII, respectively. Comparative experiments were performed using conventional CE analysis with UV detection. The results emphasise the significant improvements achieved by the new technology. Label free intrinsic imaging is a powerful technology and can be used in the analytical arena in numerous applications including the analysis of the increasingly growing field of herbal medicine.     

Determination of aristolochic acids in medicinal plants (Chinese) prepared medicine using capillary zone electrophoresis

Aristolochic acids (I and II) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end-stage renal failure. The aim of this work was to develop a method for the analysis of aristolochic acids in medicinal plant/Chinese prepared medicine (CPM) using (CZE). The buffer used was 30 mM sodium tetraborate at pH 9.5, detection was at 254 nm, applied voltage at 18 kV and the temperature was set at 25 degrees C. The effect of ionic strength, pH, and applied voltage on the separation was investigated. The precision values (relative standard deviation, RSD, %) for the relative migration time and peak area or peak height for aristolochic acids I and II were found to be less than 0.3% and between 2.6 to 4.0%, respectively. The limit of detection for aristolochic acids I and II was found to be 1.2 and 0.9 mg/L, respectively. The proposed method using pressurized liquid extraction (PLE) with CZE was used to determine the amount of aristolochic acids in medicinal plants or CPM samples with complex matrix and the results were compared with high-performance liquid chromatography (HPLC). Method precision (RSD, n = 6) was found to be less than 4% when those from applied to medicinal plants and CPM samples.     

Carbon-13 NMR spectra of some phenanthrene derivatives from Aristolochia indica and their analogues

¹³C NMR spectra of compounds related to aristolochic acid and aristololactam, the constituents of Aristolochia indica, have been studied to determine the chemical shifts and coupling constants of polysubstituted phenanthrenes. Selective ¹H decoupling and long-range couplings were utilized for the assignments. Substituent-induced chemical shifts and also the effects on coupling constants could be deduced in some cases. Anion formation was found to be particularly helpful in the interpretation of the spectra of carboxylic compounds. Shift assignments of some structurally related compounds could also be made.     

Determination of caffeoylquinic acids and flavonoids inCynara scolymus L. by high performance liquid chromatography

Summary The main phenolic compounds in dried extracts fromCynara scolymus (artichoke)—monocaffeoylquinic acids, dicaffeoylquinic acid, and flavonoids–have been separated by high-performance liquid chromatography. By use of a narrow bore C₁₈ column and an acidic mobile phase this HPLC method enabled improved separation within 31 min with significantly reduced solvent consumption compared with other methods. The method was validated to demonstrate its linearity, precision, accuracy, and robustness. Twelve commercial samples were analyzed. Monocaffeoylquinic acids were the most abundant phenolic compounds; the amounts present ranged from 0.48 to 4.24%. The amounts of dicaffeoylquinic acids and flavonoids were smaller—from 0.03 to 0.52%. The method is a good combination of efficiency and economy and should be especially useful for commercial applications.     

Pressurized liquid extraction of berberine and aristolochic acids in medicinal plants

Berberine and aristolochic acids I and II present naturally in medicinal plants were extracted using a laboratory-made pressurized liquid extraction (PLE) system in the dynamic mode. As the target analytes were present naturally in the medicinal plants, spiking was not done and comparison with ultrasonic extraction and Soxhlet extraction was performed to assess the method accuracy. The effect of temperature, volume of solvent required and particle size were investigated. Method precision (RSD, n=5) between 1.98 and 3.4% was achieved for the extraction of berberine and aristolochic acids I and II in medicinal plants and lower than 8% for lower levels of aristolochic acid II in medicinal plants.